Supplementary MaterialsS1 Fig: Anti-Gr-1 antibody treatment depletes both Ly6Chi MCs and neutrophils in uninfected mice, and stream cytometric gating technique for identifying Ly6Chi neutrophils and MCs. (B) IL-12p40-YFP reporter mice (YET40) had been uninfected (na?ve) or infected with Lp. The percentages of YFP+ DCs and MCs within the lung were quantified at 48 hours post-infection. B6 mice had been uninfected (na?ve) or infected with (Lp). Intracellular cytokine staining for IL-12p40 was performed on lung cells. Representative movement cytometry plots and graphs display the total amounts and percentages of IL-12p40-expressing neutrophils (C) within the lung at a day post-infection. (D) IL-12p40-YFP reporter mice (YET40) had been uninfected (na?ve) or infected with Lp. Representative movement cytometry plots and graphs display the total amounts and percentages of YFP-expressing neutrophils within the lung at 48 hours post-infection. YFP gates had been drawn predicated on neutrophils from B6 mice contaminated with Lp. Data demonstrated will be the pooled outcomes of 3 (A & C) or 2 (B & D) 3rd party experiments with three or four 4 contaminated mice per group per test. * can be p 0.05, ** is p 0.01, and *** is p 0.001 by unpaired t-test. NS isn’t significant.(PDF) ppat.1006309.s002.pdf (213K) GUID:?5133D9F7-556E-4C9D-9B2B-926D564A7913 S3 Fig: Neutrophils express and mRNA during pulmonary infection. B6 mice had been contaminated with and RNA Seafood was performed on lung cells 48 hours post-infection. Neutrophils had been determined by polymorphonuclear morphology within the DAPI route, and evaluation of RNA Seafood probes was performed on neutrophils (disease. Graphs displaying the total amounts of NK cells (A) and percentages of IFN+ NK cells (B) within the lungs of Lp-infected B6 or disease. Representative movement cytometry plots (A) and graphs (B) displaying the percentages and total amounts of IFN+ T cells within the lungs of B6 or Lp or uninfected (na?ve) in a day post-infection. Representative movement cytometry plots (C) and graphs (D) displaying the percentages and total amounts of IFN+ T cells within the lungs of Lp-infected B6 mice treated with isotype control (ISO) or anti-Gr-1 (-Gr-1) antibody at a day post-infection. Data demonstrated will be the pooled MK-0557 outcomes of 2 3rd party tests with 4 to 7 mice per group per test (A & B) or the pooled outcomes of 3 3rd party experiments with three or four 4 mice per group per test (C & D). NS isn’t significant by one-way ANOVA (B) or unpaired t-test (D).(PDF) ppat.1006309.s005.pdf (173K) GUID:?D517678D-567D-46DF-983E-7899565B650D S6 Fig: NKT cells and T cells produce IFN subsequent pulmonary infection, and MCs are necessary for IFN production by T cells however, not NKT cells. Graphs displaying the percentages of IFN+ NKT cells (A) and IFN+ T cells (B) within the lungs of na?ve and Lp-infected disease and B6. Representative movement cytometry plots and graphs displaying the percentages and total amounts of IFN+ NKT cells (A and B) or IFN+ T cells (C and D) within the lungs of uninfected (na?ve) B6 mice or Lp-infected B6 mice treated with isotype control (ISO) or anti-Gr-1 (-Gr-1) antibody in a day post-infection. (E) Graphs displaying the total amounts of IFN+ T cells, NK MK-0557 cells and NKT cells within the lungs of Lp-infected B6 mice treated with either isotype control antibody (ISO) or anti-Ly6G (-Ly6G) antibody, as dependant on movement Rabbit Polyclonal to UBD cytometry. Data demonstrated will be the pooled outcomes of 3 3rd party experiments with three or four 4 mice per group per test (A-D) or 2 3rd party experiments with 3 mice per group per experiment (E). MK-0557 * is p 0.05 by unpaired t-test. NS is not significant.(PDF) ppat.1006309.s007.pdf (194K) GUID:?C9EEEDD8-6549-44FE-827B-781251ADFF85 S8 Fig: Immunofluorescence microscopy reveals non-specific IFN and IL-12 MK-0557 staining in neutrophils from and mice were infected with (Lp) and immunofluorescence microscopy analysis was performed on neutrophils harvested by BAL at 48 hours post-infection stained with anti-IFN or anti-IL-12 antibodies directly conjugated to AlexaFluor488. (A) Representative images of IFN immunofluorescence (40x). (B) Representative images of IL-12 immunofluorescence (20x). Shown are the merged DAPI and AlexaFluor488 channels. In each image, a representative cell with positive fluorescence signal is outlined in a yellow box and displayed in a magnified inset.(PDF) ppat.1006309.s008.pdf (340K) GUID:?48C5482A-BDE7-406B-B4A2-58F08BAB5466 Data Availability.